IMiD® Immunomodulatory Drugs Lenalidomide and Pomalidomide Inhibit the Maturation of Megakaryocytes by Suppressing the Expression of GATA1

The IMiD® immunomodulatory drugs lenalidomide (LEN) and pomalidomide (POM) yield high response rates in patients with multiple myeloma (MM). However, the use of IMiDs is associated with neutropenia and thrombocytopenia. It has been shown before that IMiDs down-regulate the transcription factor PU.1/SPI1 leading to maturational arrest of granulocytes with accumulation of immature myeloid precursors and subsequent neutropenia (Blood. 2010; 115:605-614). However, the mechanism underlying the development of thrombocytopenia is unclear.

Here, we investigated the effects of IMiDs on megakaryopoiesis. In megakaryocytic colony formation assays IMiDs significantly (p<0.001) increased the numbers of CFU-megakaryocytes (Mk) compared to control (DMSO). The up-regulation of CFU-Mk was especially evident in medium/large CFU-Mk (3-fold increase), which arise from more primitive Mk progenitors. In accordance with the increased number of CFU-Mk, IMiDs led to a significant induction and expansion of immature Mks (CD41a⁺CD42b⁻ cells) from CD34⁺ cells cultured in a liquid megakaryopoiesis culture system. Further we were able to maintain and expand CD34⁺ cells for up to 4 month in the presence of POM. In those long term cultures we identified by multicolor flow cytometry 2 cell populations; CD45⁺34⁺33⁺11b⁺41⁺61⁺ hybrid cells (6.7%) and a more mature population of CD45⁺34⁻41⁺61⁺ cells (86.6%). The cell expansion was mediated by decreased apoptosis as well as induction of proliferation. The number of apoptotic cells significantly (p<0.05) decreased in the presence of IMiDs (apoptotic cells on day 7: 58% DMSO, 22% LEN and 6% POM; day 14: 68% DMSO, 24% LEN and 8%, POM). Further, after IMiD treatment, cell cycle analysis revealed a significant (p<0.05) increase of cells in G2/S phase (day 7: 8% DMSO, 16% LEN and 19% POM; and day 14: 7% DMSO, 14% LEN and 15% POM) suggesting that IMiDs induce proliferation.

The finding that IMiDs induce development of megakaryocytic precursors is in contrast to the clinical observation that IMiDs induce thrombocytopenia. Since previous studies showed that IMiDs are not directly toxic to bone marrow hematopoietic cells (Blood. 2005; 105:3833-40), we hypothesized that these effects might be induced by a maturational arrest of megakaryopoiesis resulting in thrombocytopenia. Therefore we studied the morphology of Mks for signs of immaturity. Giemsa staining and CD61 immunohistochemistry showed Mks developed in the presence of IMiDs were smaller in size with hypo-lobulated nuclei. 45% of cells treated with LEN, and 52% treated with POM exhibited a single nuclear lobe compared to 23% of cells treated with DMSO. We confirmed our findings by measuring the ploidy of cultured Mks by FISH. Mks (CD61-FITC-positive) were studied from each treatment (DMSO and IMiDs) and the number of nuclei and the number of centromeric region of chromosome 6 (CEP 6) signals for each Mk were recorded. CD61-FITC-negative cells were omitted from the FISH analyses. The percentage of 2N (disomic) cells was 2.5-fold higher (p=0.0221) in the IMiDs group compared to control group; in contrast the percentage of 4N and ≥8N cells (with multiple disomic nuclei) decreased 1.5 and 1.3-fold (p<0.05) in the IMiDs-treated cells compared to control treatment.

Since failure of terminal differentiation and excessive proliferation of Mks has been described in GATA1-deficient Mks (Blood. 2005; 106:1223-1231) we analyzed the expression of GATA1 at the mRNA and protein levels after treatment with IMiDs using real-time PCR, Western blot and immunofluorescence microscopy, respectively. IMiDs-treated Mks showed down-regulation of GATA1 associated with decreased expression of FOG1/ZFPM1 and NFE2 both critically interacting with GATA1. Further, previous studies suggested that polyploidy formation in Mks depends on the expression of cyclin D1 isotypes (Blood. 2007; 109:5199-5207). Indeed, IMiDs treatment caused a decrease of p21 and CCND1.

Taken together, our findings suggest that IMiDs maintain and expand early hematopoietic progenitors up to several weeks. Our data further indicate that the loss of key transcription factors such as GATA1 and CCND1 both critical for megakaryopoiesis precludes Mks from continued maturation. This might lead to maturational arrest of Mks with accumulation of immature megakaryocytic precursors and subsequent thrombocytopenia.

Lentzsch:Celgene Corp: Consultancy, Research Funding; Onyx: Consultancy; Genzyme: Consultancy; prIME Oncology: Honoraria; Imedex: Honoraria; Clinical Care Options: Honoraria.